A. MLH1:PMS1[unreadable] Yeast MutLalpha is a heterodimer of MLH1 and PMS1 that participates in a variety of DNA transactions, including DNA mismatch repair. Formation of the MutLalpha heterodimer requires that the Cterminal domains of MLH1 and PMS1 interact in a manner that is not yet fully understood. Here we investigated the interactions involved in heterodimerization. Using protein surface modification and mass spectrometry, we identified numerous lysine residues that are exposed to solvent in monomeric MLH1. Corresponding analysis of the MLH1PMS1 heterodimer revealed that three of these exposed residues, K665, K675 and K704, are no longer solvent accessible in the heterodimer, suggesting that they are within the dimer interface. We refined secondary structure predictions and sequence alignments of Cterminal residues of seven eukaryotic MutL homologs and then developed homology models for the N and Cterminal domains of MLH1. Based on this information, we presented a model for interaction of the Cterminal domains of MLH1 and PMS1.[unreadable] [unreadable] B. Calmodulin[unreadable] We have carried s out differential oxidative surface modification study on the calmodulin system which relatively well studied to validate our procedure. In differential oxidative surface mapping, the protein is subjected to hydroxyl radical induced oxidation formed via 137Cs radiolysis. Changes in the extent of oxidation are followed as a function of dose (time) and sites of oxidation are identified by tandem mass spectrometry. Calmodulin (CaM) is known to undergo conformational and functional changes on oxidation, allowing CaM to function as an oxidative stress sensor. We report the use of a novel mass spectrometrybased methodology to monitor the structure of apo and holoCaM as it undergoes conformational changes as a result of increasing amounts of oxidative damage. The kinetics of oxidation for eight peptides are followed by mass spectrometry, and 12 sites of oxidation are determined by MSMS. Changes in the pseudofirstorder rate constant of oxidation for a peptide after increasing radiation exposure reveal changes in the accessibility of the peptide to the diffusing hydroxyl radical, indicating conformational changes as a function of increased oxidative damage. For holoCaM, most sites rapidly become less exposed to hydroxyl radicals as the protein accumulates oxidative damage, indicating a closing of the hydrophobic pockets in the N and Cterminal lobes. For apoCaM, many of the sites rapidly become more exposed until they resemble the solvent accessibility of holoCaM in the native structure and then rapidly become more buried, mimicking the conformational changes of holoCaM. At the most heavily damaged points measured, the rates of oxidation for both apo and holoCam are essentially identical, suggesting the two assume similar structures. (Biophysics J.)[unreadable] [unreadable] C. Anthrax Protective Antigen[unreadable] We have continued our structural studies of the Bacillus anthracis Protective antigen and of epitopes on PA recognized by antibodies. We are studying the conformational change undergone by PA at low pH using differential oxidative surface mapping. In this procedure, the protein is subjected to hydroxyl radical induced oxidation formed via 137Cs radiolysis. Changes in the extent of oxidation are followed as a function of dose (time) and sites of oxidation are identified by tandem mass spectrometry. Oxidation of PA at neutral pH is used as a control. We are currently identifying low level oxidation sites for revision of a manuscript. We are also collaborating with S. Little (USAMRIID) on the characterizaiton of epitopes on the Bacillus anthracis protective antigen recognized by monoclonal antibodies that enhances lethal toxinmediated cytotoxicity of a murine macrophage cell line. The first monoclonal antibody studied belongs to a subset of antibodies whose Fab fragment shows neutralizing behavior while the intact antibody is enhancing. In this study, we have used epitope excision extraction procedures and chemical modification followed by mass spectrometry to characterize this conformational epitope. Based on our result, we have proposed a model that can explain the apparently conflicting Fab and intact antibody immunological properties. (Infection and Immunity, submitted)[unreadable] [unreadable] D. AntiHepatitis C E2 glycoprotein antibodies:[unreadable] We have established a collaboration with Prof. Steven Foung, Stanford University, to characterize conformational epitopes on HCV E2 glycoprotein that are recognized by neutralizing monoclonal antibodies. As part of this study, a combination of differential surface modification and mass spectrometric limited were used to provide insight into potential conformational changes within the E2 protein as a result of antibody binding. We followed up on these results using alanine mutagenesis scanning to observe the effect on antibody binding of conformational changes at nonbinding residues induced by the mutation of the residues. We observed that the E2 protein readily undergoes significant conformational changes as a result of either antibody binding or mutational scanning. This conformational lability is consistent with the known significant conformational change undergone by the protein at low pH. (J. Biol. Chem., manuscript submitted)[unreadable] [unreadable] E. Factor Xa dimer[unreadable] In a collaborative project with Prof. Barry Lentz (UNCDepartment of Biochemistry and Biophysics) and Lee Pedersen (LSB and UNC Chemistry), we have used differential surface modification and mass spectrometry to obtain data to for a model of the factor Xa dimer that shows residues buried and exposed upon dimer formation induced by soluble C6PS. Circular Dichroism also shows a drop in helix content but no change in beta upon dimer formation. Dimerization kills activity towards prothrombin but not towards tripeptide synthetic peptide substrates. These data suggest which face of Xa may be involved in dimer formation. A manuscript is in preparation.[unreadable] [unreadable] [unreadable] F. Sjogrens Syndrome[unreadable] Primary Sjogrens syndrome is a chronic inflammatory autoimmune disease which typically affects the salivary and lachrymal glands, thereby, leading to dry eyes and dry mouth. By current estimations, approximately 1% of the population (mean age of onset 4555 years and malefemale ratio of 1:9) is affected by Sjogrens syndrome, thus, making it one of the most common autoimmune rheumatic diseases. The mechanism of the development of Sjogrens syndrome is not clear, however, inflammation and lymphocyctic infiltration of the exocrine glands are a characteristic feature of the syndrome. Upon progression of the disease, the exocrine glands eventually become fibrotic, and, thus, nonfunctional. Most autoimmune rheumatic diseases are characterized by the presence of autoantibodies. The autoantibodies commonly observed in patients with Sjogrens syndrome include those against a 52 kDa RoSSA and a 48 kDa LaSSB antigen. The role of these proteins in the development of this autoimmune condition is thought to be critical for understanding the mechanism of the development of Sjogrens. To understand why these proteins become the target of an autoimmune response, a better understanding of these proteins structure and function is needed.[unreadable] We have used chemical modification of surface exposed residues (acetylation of lysines) and crosslinking experiments (DST and DTSSP) in combination with mass spectrometry to try to gain structural information of the fulllength Ro52 and LaSSB proteins. Molecular models of the structures of these proteins consistent with the mass spectrometric data have been generated.